Abstract Bacteria have been cooperating and competing for all of evolutionary time. Only recently, however, have we seriously begun to take this into account in the context of infectious disease mechanisms. The expanding ability to study bacteria in complex consortia (i.e. how they normally live) has sparked a renewed appreciation for the complexity of real bacterial populations and an examination of how they interact. Co-infection studies show that pathogen communities are more virulent, and this aligns with epidemiological reports that connect multispecies infections to worse patient outcomes. A common hypothesis is that this pathogen synergy comes about because of competition and communication between multiple species at an infection site. We hypothesize that Outer Membrane Vesicles (OMVs) serve as a major mediator of these interactions because they are known to facilitate both competition and communication between bacteria (in addition to direct virulence against host cells). We proposed a model in P. aeruginosa where OMV biogenesis is driven by the secretion and insertion of a self- produced small molecule into the outer leaflet of the outer membrane. We recently showed that this molecule could induce related species to overproduce OMVs when given at low concentration, and that those recipient species produced their own OMV cross-inducing factors. This raised the possibility that cross-species induction of OMV biogenesis could serve as a mechanism for multispecies communities to synergize for increased virulence. Guided by strong preliminary data, we will pursue two specific aims to characterize how multiple pathogens at an infection site might interact through cross-species induction of OMV biogenesis and what effects this would have on host cells: (1) We will begin by testing for small molecule-induced OMV formation using the known P. aeruginosa OMV-inducing compound PQS against a broad panel of important human pathogens likely to encounter each other at an infection site. Our established methods have already identified several such interactions among the ?-proteobacteria. We will then test whether actually growing species together (in contrast to using monocultures with exogenous compounds as above) will result in increased OMV production for the community. Preliminary results show that this is true for our pilot pairing of P. aeruginosa + E. coli. (2) OMVs from mono- vs. co-culture will be tested for their cytotoxic potential against macrophage and lung epithelial cells as well as their ability to degrade or sequester antibiotics (another disease-related function of OMVs). Preliminary results with P. aeruginosa + E. coli co-culture OMVs show that cytotoxicity and induction of apoptosis are both increased over monoculture OMVs, demonstrating feasibility of the approach and providing support for the hypothesis that cross-species OMV induction may contribute to pathogen synergy. Understanding pathogen interactions at infection sites is critical to understanding why multispecies infections lead to more severe disease. Given the ubiquity of OMV production among Gram-negative organisms, the insights gained from this study promise to broadly impact our understanding of multi-species pathogenesis.